PROJECT SUMMARY Intermediate filaments (IFs) are a fundamental fibrous component of the cytoskeleton within cells. Mutations in IF proteins cause or predispose humans to more than 80 diseases, meaning IFs have an essential role in human health and disease. Moreover, some IFs have been linked to proliferation and metastasis of cancers. Examples of IFs include keratins, vimentin, desmin, neurofilaments, and lamins. To fully correlate genotype with clinical phenotype for IF-based diseases, it is important to understand how mutations alter the three-dimensional protein structure of IF proteins and the filaments they assemble into. Currently, the atomic resolution basis for how IF proteins assemble into mature 10-nm IFs is not known and this represents one of the most critical unmet needs in IF biology. What is known from multiple biophysical studies is that IFs share a common coiled-coil/helical rod domain that is divided into four helical regions: denoted helix 1A, 1B, 2A, or 2B. This central rod domain is flanked by variable N-terminal head and C-terminal tail domains. This proposal aims to address a deficiency in our understanding of the atomic resolution basis for IF protein assembly into filaments. In particular, we focus on an anchoring knob-hydrophobic pocket IF assembly mechanism newly discovered in our lab. This discovery was made from two x-ray crystal structures of keratin 1/10 helix 1B tetrameric complexes ? the IF tetramer is considered the building block for higher- order filament packing. These structures raised several questions that remain unclear: (1) does the knob- pocket mechanism regulate the rate and/or the length of IF assembly; (2) is the knob-pocket mechanism conserved across the six types of IFs; (3) which residues in the knob and pocket are most critical for the interaction; (4) how do mutants of the knob or pocket alter IF assembly; and (5) can the knob-pocket mechanism be targeted with peptides or small-molecules to disrupt IF assembly. We believe focusing our studies on these important questions will advance our mechanistic understanding of IF assembly. In this project, we examine in depth the biochemical and structural properties of the anchoring knob- hydrophobic pocket IF assembly mechanism identified our laboratory. In Aim 1 we will use negative-stain electron microscopy to analyze wild-type and mutant IFs to understand how the loss of the knob-pocket interaction affects the rate and length of filament formation. Multiple IF systems will be evaluated to establish the degree of conservation of this mechanism across IFs. In Aim 2 we will selectively mutate hydrophobic pocket residues to determine which pocket residues are most critical to knob binding. Then, we will study whether knob peptides can bind to the pocket and prevent IF assembly. Accomplishing these aims will provide novel insight into how the knob-pocket mechanism governs IF assembly and establish a foundation for developing targeted therapies of IFs through their assembly mechanisms.